Comparing the hierarchy of inter- and intra-species interactions with population dynamics of wine yeast cocultures.

In winemaking, the development of new fermentation strategies, such as the use of mixed starter cultures with Saccharomyces cerevisiae (Sc) yeast and non-Saccharomyces (NS) species, requires a better understanding of how yeasts interact, especially at the beginning of fermentation. Despite the growing knowledge on interactions between Sc and NS, few data are available on the interactions between different species of NS. It is furthermore still unclear whether interactions are primarily driven by generic differences between yeast species or whether individual strains are the evolutionarily relevant unit for biotic interactions. This study aimed at acquiring knowledge of the relevance of species and strain in the population dynamics of cocultures between five yeast species: Hanseniaspora uvarum, Lachancea thermotolerans, Starmerella bacillaris, Torulaspora delbrueckii and Sc. We performed cocultures between 15 strains in synthetic grape must and monitored growth in microplates. Both positive and negative interactions were identified. Based on an interaction index, our results showed that the population dynamics seemed mainly driven by the two species involved. Strain level was more relevant in modulating the strength of the interactions. This study provides fundamental insights into the microbial dynamics in early fermentation and contribute to the understanding of more complex consortia encompassing multiple yeasts trains.

Impact of Leavening Agent and Wheat Variety on Bread Organoleptic and Nutritional Quality.

Leavened bread can be made with different wheat varieties and leavening agents. Several studies have now demonstrated that each of these factors can play a role in bread quality. However, their relative impact in artisanal bread making remains to be elucidated. Here, we assessed the impact of two wheat varieties as well as the impact of sourdoughs and yeasts on multiple components of bread organoleptic and nutritional quality. Using a participatory research approach including scientists and bakers, we compared breads leavened with three different sourdoughs and three different commercial yeasts as well as a mix of sourdough and yeast. Breads were made from two wheat varieties commonly used in organic farming: the variety "Renan" and the landrace "Barbu". Except for bread minerals contents that mostly depended on wheat variety, bread quality was mostly driven by the fermenting agent. Sourdough breads had lower sugar and organic acids contents. These differences were mostly attributable to lower amounts of maltose and malate. They also had a higher proportion of soluble proteins than yeast breads, with specific aroma profiles. Finally, their aroma profiles were specific and more diverse compared to yeast breads. Interestingly, we also found significant nutritional and organoleptic quality differences between sourdough breads. These results highlight the value of sourdough bread and the role of sourdough microbial diversity in bread nutritional and organoleptic quality.

Focus on putative serine carboxypeptidase-like acyltransferases in grapevine.

Grapevine (Vitis vinifera L.) berry synthesizes and accumulates a large array of phenolic compounds (e.g. flavonoids and hydroxycinnamic acid derivatives), some of which result from acylation mechanisms. In grapevine, the genes encoding enzymes responsible for such acylation are largely unknown. Enzymes classified as serine carboxypeptidases (SCPs), able to transfer acyl moieties from a glucose ester, have previously been characterized in plants, and named serine carboxypeptidase-like acyltransferases (SCL-ATs). We performed genome-wide identification of SCP sequences in V. vinifera. Phylogenetic analysis revealed that only 12 grapevine SCPs, grouped in clade IA with previously characterized SCPL-AT could have an acylation function. Interestingly, seven putative SCP-ATs are grouped in a 400 kb cluster in chromosome 3. The expression level of putative SCPL-ATs has been evaluated at key stages of grape berry development in the main tissues and compared with the content of acylated phenolic compounds in the corresponding samples. The expression levels of VvGAT1 and VvGAT2 and that of VvSCP5 were increased in hairy-roots overexpressing transcription factors inducing the biosynthesis of proanthocyanidins and anthocyanins, respectively. These findings open the way for the functional characterization of the identified putative SCPL-AT from grapevine.

Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, ß-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites.

ABCC1, an ATP binding cassette protein from grape berry, transports anthocyanidin 3-O-Glucosides.

Accumulation of anthocyanins in the exocarp of red grapevine (Vitis vinifera) cultivars is one of several events that characterize the onset of grape berry ripening (véraison). Despite our thorough understanding of anthocyanin biosynthesis and regulation, little is known about the molecular aspects of their transport. The participation of ATP binding cassette (ABC) proteins in vacuolar anthocyanin transport has long been a matter of debate. Here, we present biochemical evidence that an ABC protein, ABCC1, localizes to the tonoplast and is involved in the transport of glucosylated anthocyanidins. ABCC1 is expressed in the exocarp throughout berry development and ripening, with a significant increase at véraison (i.e., the onset of ripening). Transport experiments using microsomes isolated from ABCC1-expressing yeast cells showed that ABCC1 transports malvidin 3-O-glucoside. The transport strictly depends on the presence of GSH, which is cotransported with the anthocyanins and is sensitive to inhibitors of ABC proteins. By exposing anthocyanin-producing grapevine root cultures to buthionine sulphoximine, which reduced GSH levels, a decrease in anthocyanin concentration is observed. In conclusion, we provide evidence that ABCC1 acts as an anthocyanin transporter that depends on GSH without the formation of an anthocyanin-GSH conjugate.

Proline-rich salivary proteins have extended conformations.

Three basic proline-rich salivary proteins have been produced through the recombinant route. IB5 is a small basic proline-rich protein that is involved in the binding of plant tannins in the oral cavity. II-1 is a larger protein with a closely related backbone; it is glycosylated, and it is also able to bind plant tannins. II-1 ng has the same polypeptidic backbone as II-1, but it is not glycosylated. Small angle x-ray scattering experiments on dilute solutions of these proteins confirm that they are intrinsically disordered. IB5 and II-1 ng can be described through a chain model including a persistence length and cross section. The measured radii of gyration (Rg=27.9 and 41.0+/-1 A respectively) and largest distances (rmax=110 and 155+/-10 A respectively) show that their average conformations are rather extended. The length of the statistical segment (twice the persistence length) is b=30 A, which is larger than the usual value (18 A-20 A) for unstructured polypeptide chains. These characteristics are presumably related to the presence of polyproline helices within the polypeptidic backbones. For both proteins, the radius of gyration of the chain cross-section is Rc=2.7+/-0.2A. The glycosylated protein II-1 has similar conformations but the presence of large polyoside sidegroups yields the structure of a branched macromolecule with the same hydrophobic backbone and hydrophilic branches. It is proposed that the unusually extended conformations of these proteins in solution facilitate the capture of plant tannins in the oral cavity.

Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.

Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.